The Aequorea victoria green fluorescent protein (GFP), along with its various homologues and mutants (Shaner, 2005; Shimomura, 1979), has enabled live-cell fluorescence imaging of recombinant fusion proteins to become a popular and widely-accessible technique in cell-biology research (Tsien, 1998; Zhang, 2002). The defining feature of Aequorea GFP is its ability to autonomously generate a green fluorophore within the confines of its distinctive β-barrel structure (Shimomura, 1979; Yang, 1996; Ormo, 1996). The chromophore of GFP is post-translationally and autonomously generated, through a stepwise process that involves a main-chain cyclization (Gly67 N to Ser65 C), a dehydration (Ser65 C—N), and an oxidation (Tyr66 Ca—C) that effectively conjugates the phenolic side chain of tyrosine 66 to a five-membered ring heterocycle formed from the main-chain atoms of serine 65, tyrosine 66, and glycine 67 (FIG. 1). In the ground state of the wild-type GFP, the chromophore exists as a mixture of neutral phenol (maximum absorbance at 395 nm) and anionic phenolate forms (maximum absorbance at 475 nm). In the excited state, the neutral phenol form deprotonates to form the phenolic anion; therefore, only a single fluorescence emission peak (maximum fluorescence at 504 nm) is observed.
The steric, electrostatic, and hydrogen-bonding environment imposed upon the chromophore by the surrounding residues strongly influences the fluorescence properties. The GFP chromophore has proven remarkably amenable to genetic modification of both its covalent structure and its local environment, and this tolerance has been exploited for the creation of wavelength-shifted variants (Tsien, 1998). Aequorea GFP variants (Shaner, 2005) have been engineered with altered colors, brightness, photostability, ion-sensitivity (Hanson, 2002), and photoswitching properties (Lukyanov, 2005). Amino-acid substitutions at position 65 and at several other residues in the immediate vicinity of the chromophore (e.g., position 203) have resulted in GFP variants (i.e., enhanced GFP (EGFP) with maximum fluorescence at 510 nm). A particularly important class of useful variants that have resulted from such efforts is the yellow fluorescent proteins (YFPs) that are defined by the Thr203Tyr mutation (Ormo, 1996) and an emission peak that is ˜25 nm red-shifted from the wild type emission peak of ˜504-509 nm. However, at present, there is no known report of an Aequorea GFP mutant with a tyrosine-derived chromophore and fluorescence that is blue-shifted relative to the wild-type protein (i.e., it has a maximum fluorescence that is less than 504 nm).
The term “cyan fluorescent protein”, or “CFP”, is generally reserved for any GFP homologue with maximum fluorescence emission between approximately 470 nm and 495 nm. To date, substitutions of tyrosine 66 to other aromatic amino acids have proved to be the only approach for blue-shifting the fluorescence emission relative to the wild-type protein, in order to produce a CFP. For example, the widely used Aequorea GFP-derived CFP known as avCFP (also commonly known as ECFP or CFP) was engineered by replacing Tyr66 of Aequorea GFP with a tryptophan, to give an indole-containing chromophore (FIG. 1) (Heim, 1994) that had an emission peak in the cyan region (˜480 nm) of the visible spectrum. Although the original Tyr66Trp mutant of Aequorea GFP was only weakly fluorescent, efforts to improve the brightness yielded the widely used variant ECFP (Heim, 1994; Miyawaki, 1997) and more recently Cerulean (Rizzo, 2004) and CyPet (Nguyen, 2005). While avCFP has been proven as a useful fluorophore in multicolor labeling applications, and as the preferred Forster resonance energy transfer (FRET) donor to a YFP acceptor, its spectral properties limit its utility in some applications. Specifically, avCFP is relatively dim, has broad excitation and emission peaks (FIG. 2), and has a multi-exponential fluorescence lifetime. The multi-exponential fluorescence lifetime of avCFP complicates the use of this protein in fluorescence lifetime imaging (FLIM) applications. Some limitations have been partially addressed in the newer variants; Cerulean is twofold brighter and has a more homogenous fluorescence lifetime (Rizzo, 2004), while CyPet exhibits high FRET to the YFP variant YPet (Nguyen, 2005). However, despite these improvements, Cerulean and CyPet remain limited by fluorescent brightness that is less than 50% of the popular YFP variant Citrine (Shaner, 2005) and that is inferior to EGFP, and by fluorescence lifetimes that are poorly fit as single-exponentials, and a very broad fluorescence emission relative to other popular variants (FIG. 2) (Rizzo, 2004).
Thus, there is a need in the art for a fluorescent protein which mitigates the difficulties of the prior art.